Molecular dissection of the genes involved in A. fumigatus spore color synthesis and their role in virulence. Aspergillus is one of the most common fungal pathogens affecting neutropenic patients and other types of immunocompromised individuals such as those with Chronic Granulomatous Disease of Childhood. Among a dozen species of Aspergillus reported to cause infection in humans, A. fumigatus is the most common species reported to cause invasive aspergillosis. All Aspergillus species propagate by conidia (spores), which humans encounter daily through inhalation. We have focused our attention on the molecular genetic aspects of conidial pigment biosynthesis since pigment is one of the visible components of the wall that protect conidia. In the previous years we have characterized six developmentally associated genes involved in pentaketide melanin synthesis which are clustered within a 19kb fragment of A. fumigatus genomic DNA. Furthermore, we have shown that the conidial pigment synthetic pathway plays an important role in pathogenesis. In 2001, we identified the product of one of the six clustered genes, AYG1, a novel protein. Ayg1p, shortens the length of the polyketide carbon skeleton from a heptaketide to pentaketide. This year we have attempted the identification of a regulator for the developmentally associated conidial pigment gene expression. We have also continued the characterization of a gene responsible for thermophilism of the species. A mutant that grows at 42C but not at 48C was isolated by treating A. fumigatus conidia with a mild mutagen. The mutant was complemented with a genomic library to isolate a gene (tht1) that is associated with the thermophilic phenotype. The tht1 gene is present among the members of the A. fumigatus group (i.e. A. fumigatus, A. fischeri, A. fenneliae) but absent in other aspergilli less frequently isolated from invasive pulmonary aspergillosis such as A. flavus and A. nidulans. The tht1 gene was disrupted in a clinical strain, B-5233, and the disruptant was found to have lost the ability to grow at 48C. Blast searches with the tht1 nucleotide and amino acid sequences have not revealed any homologous sequences. The function of tht1 remains unknown at this writing. Complementation of the tht1 mutant with the wild type tht1 gene restored the thermophilic phenotype in the mutant. The tht1 cDNA was observed to possess one intron and three upstream ORFs at the 5' region. Nothern blot analysis indicated that the tht1 gene is expressed at low levels and the tht1 mRNA is present in cultures grown at 37C as well as at 48C. Cultures grown at 37C and 48C showed no significant difference in the amount of the Tht1 protein. Deletion of the tht1 gene had no effect on virulence in mice as expected since the mutant can grow at 37-39C as well as the wild type strain. We, therefore,concluded that tht1 is essential for growth of A. fumigatus at high temperatures and could only be important for the pathogenesis in seriously febrile immunosuppressed patients.